The following proposal is designed to provide the principal investigator Frederick Racke with the necessary scientific experience to allow for a successful transition to an independent clinician scientist. Dr. Racke received his M.D./Ph.D. degrees at Case Western Reserve University School of Medicine. During his graduate studies in the department of Physiology and Biophysics, he acquired expertise in the study of signal transduction while working on the role of protein kinase C in the regulation of parathyroid hormone secretion in the laboratory of Edward Nemeth. Following the completion of medical school, Dr. Racke pursued residency training in the area of clinical pathology and fellowship training hematopathology at University Hospitals of Cleveland. During this training period, Dr. Racke worked in the laboratory of Adam Goldfarb, studying the role of the extracellular signal-related kinase (ERK) in phorbol ester-induced megakaryocytic differentiation of the human erythroleukemia K562 cell line. In July 1998, Dr. Racke will become a junior faculty member in the Department of Pathology at the Johns Hopkins Medical Institution. He will participate in the clinical and teaching activities of hemapathology division involving 20-25 percent of his effort. The remainder of his effort will be involved in the establishment of a scientific program under the mentorship of Dr. Chi Van Dang and Dr. Paul Bray, members of the departments of Hematology and Pathology. The aim of this program will be to acquire the necessary skills in molecular biology and the culture and manipulation of hematopoietic stem cells to allow for a gradual transition to an independent status. Specifically, the research plan revolves around defining the roles of specific isozymes of protein kinase C (PKC) in the lineage determination and differentiation of megakaryocytic cells. The central hypothesis is that specific isozymes of PKC are critical to the lineage commitment of megakaryocytic cells. Activation of these isoforms regulates the transcription of critical megakaryocyte genes required for the initiation of the megakaryocyte differentiation programming. These PKC- dependent events may or may not be part of the thrombopoietin (TPO) signaling cascade, and may work in coordination with thrombopoietin signaling. Introduction of mutants of specific PKC isoforms into models of PKC- and TPO-induced differentiation will provide the cornerstone to evaluating their role in these processes.